The goal of this proposal is to develop an injectable gel carrier that would allow simple and reproducible clinical delivery of human mesenchymal stem cells (hMSCs) into the jaw after tooth extractions. The gel environment will be tailored to provide localized signals to induce osteogenesis, maintain cell function and promote mineralization, and lead to integration with the native tissue. Within the hydrogel carrier, we plan to design 3D scaffold chemistries that support hMSC viability and proliferation (aim 1), osteogenic differentiation (aim 2), and mineralized tissue formation (aim 3). We hypothesize that three factors will be important in this design. First, we aim to tune the macroscopic hydrogel properties including water content, mesh size, and degradation rate to support 3D hMSC culture. Second, we propose that the composition of the extracellular hydrogel environment and localized presentation of osteogenic factors will induce hMSC differentiation to osteoblasts. Third, we will manipulate the gel degradation mechanism (i.e., hydrolytic vs proteolytic) and rate, as well as introduce mineralization nucleators, to facilitate neotissue evolution. The experimental approach for each aim will be to photoencapsulate hMSCs in poly(ethylene glycol) (PEG)-based hydrogels. BRDU incorporation and gene expression with time, as determined by real time RT-PCR, immunostaining, and in situ hybrization, will be used to assess hMSC cell proliferation and differentiation. Functional activity of differentiated hMSCs will be assessed by measuring alkaline phosphatase activity, calcium deposition, and extracellular matrix formation. Effects of gel chemistry, especially the introduction of osteogenic epitopes and mineralization nucleators, on hMSC function will be screened by culturing cell-laden hydrogels in vitro. The gel provides a three dimensional environment that is easily controlled to mimic critical aspects of bone extracellular space during fracture healing. Results from these aims will be used to identify hydrogel formulations that permit hMSC function, promote osteogenic differentiation, and facilitate mineralized tissue formation. These in vitro results will then provide the foundation to select formulations for testing in an animal model of bone regeneration in a critical-sized rat calvaria defect (aim 4).